In recent years, technical improvements in epifluorescence microscopy have centered on increasing the contrast between fluorescently labeled specimen components and background. As a result, many thousands of fluorescent probes have been developed to provide a means of labeling many different cellular, subcellular and molecular components of a biological specimen. In addition, the large spectral range of available fluorophores allows simultaneous imaging of different components. In order to image different components of a specimen, the different components are labeled with fluorophores that fluoresce at different wavelengths, but each fluorophore is excited with an excitation beam of a different wavelength. As a result, those working in epifluorescence microscopy have directed much attention to developing efficient and cost effective ways of superimposing the excitation beams so that the beams travel along the same path through the objective lens into the specimen. Gas-tube lasers and stacks of dichroic mirror are two systems that have been consider for producing superimposed excitation beams. Gas-tube lasers typically emit a single beam of light composed of several distinct wavelengths and a stack of dichroic mirrors can be used to combine excitation beams that emanate from different light sources. FIG. 1 shows an example of two dichroic mirrors 102 and 104 stacked to superimpose three excitation beams of different wavelengths λ1, λ2 and λ3 represented by patterned lines 106-108, respectively. Dichroic mirror 102 transmits the beam 106 and reflects the beam 107 and dichroic mirror 104 transmits the beams 106 and 107 and reflects the beam 108 to form a superimposed beam 110 composed of all three wavelengths.
However, typical gas-tube lasers are large, cost prohibitive, inefficient, and unstable. Gas-tube lasers also have short lifetimes and emit light over a very limited range of wavelengths. On the other hand, although the dichroic mirror-based approach is versatile, each time an excitation beam is added to the superimposed beam, a separate dichroic mirror is added to the stack which leads to substantial inefficiency as the non-negligible losses accumulate. For instance, as shown in FIG. 1, the beam 106 already passes through the two dichroic mirrors 102 and 104. Addition of a third dichroic mirror to the stack to reflect a fourth wavelength and transmit the wavelengths λ1, λ2 and λ3 would further attenuate the beam 106. In addition, neither gas-tube lasers nor dichroic mirror stacks provide switching between the different excitation beams on the sub-millisecond time scale or faster. With the dichroic mirror-based approach, it is possible to place shutters in the path of each beam input to the stack. However, each shutter adds substantial cost to the instrument and shutters are not able to achieve the desired sub-millisecond switching speeds between different excitation beams. For the above described reasons, engineers, scientists, and fluorescent microscope manufacturers continue to seek fast, efficient, and cost effective systems for placing excitation beams along the same path.